We have been studying development using the bacterium Myxcococcus xanthus as a model system. This bacterium is interesting because it has a complex life cycle involving cellular aggregation and fruiting body formation. In the last project period, we discovered that the frizzy (frz) genes, which are required for development, control directional movement of the cells and are similar in many of their functions to the chemotaxis genes of the enteric bacteria. We plan to focus our effort during the next project period on further characterization of the frizzy sensory transduction system and its interaction with the still unidentified motility apparatus. Our specific objectives are: (a) To purify and characterize the frz gene products and to prepare antibodies to each of the proteins. Protein-protein interactions will be examined in order to gain insight into the functioning of the signal transduction system. This will involve both a biochemical approach, in which interacting proteins are identified by crosslinking or by co- immunoprecipitation, and a genetic approach, in which allele-specific suppressors identify gene products which interact. It is hoped that these approaches will help identify the putative membrane-bound receptor which is thought to interact with FrzCD, the methyl-accepting taxis protein as well as the switch for the motor involved in gliding motility which is thought to interact with FrzE. (b) To identify chemicals/signals that stimulate methylation and demethylation of FrzCD and which alter the directional motility of M. xanthus. The methylation state of FrzCD can be used as an assay to monitor what is being perceived by intact cells, both vegetative and developmental. The behavior of cells to these chemicals/signals will then be studied by time-lapse video microscopy. (c) To study the regulation of expression of the frz genes. We plan to identify the transcriptional promoters by primer extension and to characterize the RNA by Northern blot hybridizations. The DNA-binding activity of purified FrzCD will be examined because this protein has been shown to be necessary for full expression of frzA and frzB. We also plan to analyze the DNA region of FrZA with site-directed mutagenesis and to study the effects of overexpression of cloned frz genes on feedback regulation of transcription. This project is health-related in that it explores fundamental processes of sensory transduction in a model bacterial system which may have application in understanding of similar processes in higher cells.